A number of immunomodulatory therapeutics increase the risk of disease associated with latent herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), a member of the lymphocryptovirus (LCV) family that infects humans. The diseases associated with loss of immunity to these viruses can have major impacts on patients as well as on the commercial viability of the immunomodulatory therapeutics. In an effort to develop non-clinical methods for measuring effects on anti-viral immunity, we have developed an interferon (IFN)-g enzymelinked immunosorbent spot (ELISPOT) assay to quantify the number of CMV or LCV-reactive Tcells in peripheral blood of cynomolgus macaques. After optimization of various parameters, the IFN-g ELISPOT assay was characterized for specificity, intra-assay, monkey-to-monkey, and longitudinal variability and sensitivity to immunosuppression. The results show that nearly all animals have detectable responses against both CMV and LCV and responses were derived from T-cells specific to the virus of interest. Analyses of variability show assay reproducibility ( 23% CV), and that variability over time in anti-viral responses in individual animals (larger for LCV than for CMV) was $2-fold in most animals over a 3-month time period, which is predicted to allow for detection of drug-induced changes when using group sizes typical of non-clinical studies. In addition, the IFN-g ELISPOT assay was capable of detecting decreases in the numbers of CMV and LCV reactive T-cells induced by immunosuppressive drugs in vitro. This assay may allow for non-clinical assessment of the effects of immunomodulatory therapeutics on anti-viral T-cell immunity in monkeys, and may help determine if therapeutics increase the risk of reactivating latent viral infections.

Lymphocryptoviruses such as Epstein-Barr virus (EBV) cause persistent infections in human and non-human primates, and suppression of the immune system can increase the risk of lymphocryptovirus (LCV)-associated tumor development in both human and non-human primates. To enable LCV infection as a non-clinical model to study effects of therapeutics on EBV immunity, we determined the genomic DNA sequence of the LCV from cynomolgus macaque, a species commonly used for non-clinical testing. Comparison to rhesus macaque LCV and human EBV sequences indicates that LCV from the cynomolgus macaque has the same genomic arrangement and a high degree of similarity in most genes, especially with rhesus macaque LCV. Genes showing lower similarity were those encoding proteins involved in latency and/or tumor promotion or immune evasion. The genomic sequence of LCV from cynomolgus macaque should aid the development of non-clinical tools for identifying therapeutics that impact LCV immunity and carry potential lymphoma risk.

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